Increasing tendency of incidence of deep fungal infections has been seen as the number of compromised patients increases, and therefore, effective therapeutic agents have been desired. Currently, only five antifungal agents for deep fungal infections have been launched in the market in Japan. Among them, three agents are azole-type agents (miconazole, fluconazole and itraconazole). Fluconazole, a most typical agent, has only a fungistatic action. Moreover, with increase of amount of the agent used, appearance of resistant fungi is concerned. Amphotericin B, a polyene antibiotic having a potent fungicidal effect, is highly toxic, and the agent cannot be always used safely. From these reasons, antifungal treatment of patients with deep fungal infections often results in a poor satisfactory level, and thus demands for novel fungicidal and fungiselective agents are urgent.
A cell wall which characteristically exists in fungal cells is an attractive target from a viewpoint of selectivity. In yeast, for example, major saccharide polymers constituting the cell wall include (1,3)-β-glucan, (1,6)-β-glucan, chitin and mannan. Among synthetic pathways of these saccharide polymers, the synthetic pathway of mannan commonly exists in animal cells and fungal cells and each biosynthetic pathway has high commonness, and therefore, it is considered generally difficult, although not absolutely impossible, to find a target specific to fungi. In the synthetic pathways of (1,3)-β-glucan and that of chitin, existence of enzymes specific to fungi and essential for their growth, such as the FKS gene group and the CHS gene group, has been elucidated, and research and development of antifungal agents targeting the enzymes are being conducted. Thus, candin antifungal agents having a (1,3)-β-glucan inhibitory action have been practically developed. The (1,6)-β-glucan synthetic pathway is also considered as specific to fungi, and existence of enzymes believed to be essential for growth of fungi has been elucidated based on results of genetic analyses. However, no assay system at an enzyme level has been established, and accordingly, no inhibitor against these enzymes has been reported. For this reason, no antifungal agent inhibiting this synthetic pathway has been known to date.
In general, proteins collectively called as GPI-anchored proteins extensively exist in eukaryotic cells. These are fixed on cell membranes via GPI anchors (Ferguson, M. A., et al., Ann. Rev. Biochem., 57, pp.285-320, 1988). Each GPI-anchored protein has relatively hydrophobic signal peptide regions at both ends of the N- and C-terminals. These signals are cleaved by post-translational modification, and with addition of a GPI anchor to the C-terminus, the protein is fixed on the ER (rough endoplasmic reticulum) membrane. Then, the GPI-anchored protein fixed on the ER membrane is transported on the membrane, and then further fixed on the cell membrane (Ferguson, M. A., et al., Ann. Rev. Biochem., 57, pp.285-320, 1988; Lu, C. F., et al., Mol. Cell Biol., 14, pp.4825-4833, 1994).
In Saccharomyces cerevisiae, a part of the GPI anchor is further cleaved from some of the GPI-anchored proteins fixed on the cell membrane, and then the protein is further fixed on the cell wall via (1,6)-β-glucan as an anchor (Lu, C. F., et al., Mol. Cell Biol., 14, pp.4825-4833, 1994; Kollar, R., et al., J. Biol. Chem., 272, pp.17762-17775, 1997).
This means that, in Saccharomyces cerevisiae, two kinds of GPI-anchored proteins exist; one is fixed on the cell membrane and the other in the cell wall. This difference in localization is expected to be regulated by the difference in the signal peptide at the C-terminus (Hamada, K., et al., Mol. Gen. Genet., 258, pp.53-59, 1998; Caro, L., Yeast, 13, pp.1477-1489, 1997). Recently, creation of an yeast having an arbitrary exogenous protein fixed on the cell wall (arming yeast) was reported by utilizing the localization mechanism of proteins on cell walls (Varrt, J. M. V. D., et al., Appl. Environ. Microbiol., 63, pp.615-620, 1997; Murai, T., et al., Appl. Environ. Microbiol., 63, pp.1362-1366, 1997).
As described above, (1,6)-β-glucan has a function as an anchor for fixing the GPI-anchored proteins on the cell walls, and analytical results so far obtained reveal that, when biosynthesis of (1,6)-β-glucan is inhibited by gene disruption, these proteins are extracellularly released (Lu, C. F., et al., Mol. Cell Biol., 14, pp.4825-4833, 1994; Lu, C. F., et al., J. Cell. Biol., 128, pp.333-340, 1995).
Recently, Tsuchiya et al. reported construction of an expression system of a reporter protein bound with staphylococcus cell wall peptide glycan (The Pharmaceutical Society of Japan, The 120th Annual Meeting, Abstracts 2, p.153, Lecture No. 30 [PB] 15-71). This system comprises cephalosporinase as a reporter protein anchored on a cell wall of gram-positive bacterium. However, application of this system has not been clarified, and the publication neither suggests nor teaches that the system can be used for screening of an agent acting on a cell wall.
Further, no method has been known so far as a method for conveniently and accurately estimating targeting sites of cell wall-acting agents, except a method for screening agents relating to cell walls such as (1,3)-β-glucan and chitin. A method for screening an agent acting on a particular targeting site cannot be applied for screening of agents acting on the other targeting site.